Potatoes with increased protein content

ABSTRACT

The invention relates to the breeding and selection of potatoes. The invention provides a potato plant or part derived thereof having at least one amf-allele said potato plant or part further provided with an increased capacity to store a protein as characterized by an increased protein content of its tubers. Furthermore, the invention provides a method for breeding and selecting a potato with an increased capacity to store a protein comprising crossing a first parent potato with at least one amf-allele with a second parent potato without an amf-allele, and selecting progeny for the presence of at least one amf-allele with a protein content of its tubers higher than detected in said first parent or said second parent.

This application is the U.S. National Phase of International ApplicationNumber PCT/NL2003/000851 filed on 2 Dec. 2003, which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The invention relates to the breeding and selection of potatoes.

Apart from being an important staple food, potato is classically the rawmaterial for industrial production of starch from potato tubers.Furthermore, these days, the industrial harvest of potato protein frompotato tubers, earlier seen as a quantité négligeable attracts moreattention considering the increased value given to vegetable proteinsources, for example for animal food, if only as a side productaccompanying starch production. Where, chemically, potato starch inpotato tubers essentially consists of two components: amylopectin andamylose in a proportion of approximately 80% to 20%, potato proteins intubers essentially consist of protease inhibitors that help protect thetuber against disease such as parasite infestations or fungal orbacterial rot and storage proteins such as patatin in a proportion ofapproximately 60% to 40%.

For various reasons, starch producers prefer potatoes with differentratios of amylopectin and amylose. An earlier induced gene mutation inpotatoes that affects the synthesis of the enzyme granule bound starchsynthase (GBSS), and the subsequent molecular cloning of this gene(Hovenkanp-Hermelink et al., 1987, Theor. Appl. Genet. 75:217-221;Visser et al., 1989, Plant Science 64:185-192) has opened possibilitiesfor altering the starch composition of potatoes—either throughestablished breeding methods or through modern techniques of geneticmanipulation.

The GBSS mutation in potato is similar to the so-called waxy (wx)mutation in maize and prevents the production of amylose, whenexpression or specific function of the GBSS protein is absent.Therefore, this mutation has been designated as amylose-free (amf)mutant of potato. Herein, the amf-gene mutation stands for amodification of the GBSS-gene that leads to a complete functional lossof GBSS-activity, notwithstanding that GBSS-like gene products, withoutthe specific activity, may still be expressed from the gene'stranscripts in question, whereby the Amf-gene stands for a gene fromwhich gene products with GBSS-activity can still be obtained. Theamf-gene character is determined by a monogenic mendelian recessivegene, the phenotype of which can be detected in various plant parts suchas columella cells of root tips, tubers, plastids in the stomatal guardcells and in microspores (Jacobsen et al., 1989, Euphytica 44:43-48).When these parts are stained with a potassium iodine solution (Lugol),starch is stained red in mutants and dark blue in the wild type.

Unlike many other phenotypic genetic markers, the mutated orfunctionally deleted GBBS- or amf-gene offers certain special advantagesfor genetic analysis as well as for breeding. For example, the progenycan be classified at a very early seedling stage as well as in adultplants, through pollen staining, homo- and heterozygotes can beunambiguously classified: the dosages 2-4 of the mutant allele in atetraploid can be easily detected through the ratios 5:1, 1:1 and 0:1 instained pollen samples; different types of 2n-gametes in diploid clonescan be detected and their influence on the phenotype and genotype oftetraploid from 4x*2x crosses can be predicted.

Prospects of using the material in conventional as well as in analyticbreeding of potato have since the development of the amf-gene potatomutant of Hovenkamp-Hermelink been opened. A disadvantage for breedingis the recessive nature of amf, which complicates the combination ofthis character with other agronomic traits at the tetraploid level.

Therefore, the analytic breeding method advocated by Chase (1963, J.Genet. Cytol. 5:359-364), which involves breeding of potato at thediploid level and returning to the tetraploid condition through the useof 2n-gametes, could be of considerable value for breedingamf-varieties. The aim of such investigations are at least two fold: a.to combine amfamf and Amfamf genotypes with that of 2n-gamete formation,and b. to create fertile, nulliplex clones as basic material forbreeding amylose-free potatoes. On the other hand, development ofsuitable diploid material that produces high frequencies of 2n-pollenand 2-eggs would also open the way for unilateral and bilateral sexualpolyploidization (Mendiburu and Peloquin, 1976, Theor. Appl. Genet.48:137-143). Such diploid breeding material may be homozygous (amfamf)or heterozygous (Amfamf), because in both cases selection can be carriedout based on pollen phenotype.

SUMMARY OF THE INVENTION

The invention relates to the breeding and selection of potatoes.Surprisingly, it was found herein that potatoes with at least oneamf-allele background have a distinct phenotypic advantage when comparedwith potatoes having a similar genetic background lacking the amf-gene.One such advantage relates to protein content. Genotypes that arenulliplex for the Amf-allele, i.e. for diploid plants the amfamf (aa)and for tetraploid plants the amfamfamfamf (aaaa) genotypes display saidadvantage even stronger.

The invention provides a potato plant or part derived thereof (such as acell, a protoplast, a tuber, an embryo, a seed or an explant) having atleast one amf-gene said potato plant or part further provided with anincreased capacity to store a protein (herein also identified as a highprotein potato) as characterized by a total raw protein content of itstubers (preferably as determined in the potato juice derived thereof) atleast 1-9% m/m, more preferred at least 2.3% m/m, most preferred atleast 2.7% m/m. Within a potato homozygous for the amf-allele asprovided herein, i.e. an amylose-free high protein potato, suchincreased capacity to store protein is most fully developed. Theinventors have gathered the surprising insight that depriving a potatoof GBSS-activity allows for increasing protein storage in said potato,provided it has the genetic capacity to produce increased, or at leastsufficient, amounts of said protein. Potatoes comprising an amf-allelehave essentially higher protein storage capacity than potatoes ofotherwise similar genetic background having no amf-gene. Potatoeshomozygous for the amf-allele are, speaking from the viewpoint ofprotein storage, preferred.

Originally, the amf-mutation was induced in a monohaploid which had beenselected only for flowering (Hovenkamp-Hermelink et al., 1987, ibid) butnot for fertility and agronomic characters. Therefore, in order toincorporate this recessive mutant in other potatoes the inventorscrossed the diploid genotype derived from the monoploid mutant clonewith agronomically more desirable clones which, however, have the wildtype of the Amf gene. As a first step in this process, fertile diploidsthat are homozygous for the mutant character (amfamf), were produced.When these diploids are somatically doubled through in vitroadventitious shoot regeneration, the resulting tetraploids proved to beless fertile (both male and female). However the 4x plants obtainedthrough meiotic doubling—using 4x×2x crosses—gave rise to fertilenulliplex tetraploids. Thus, in spite of high levels of sterility andexpression of lethal factors in the initial stages, more fertile andvigorous diploid and tetraploid breeding material were created with thedesired amf-genotypes. Availability of vigorous, fertile and agronomicaluseful tetraploid genotypes than led to conventional breeding ofamf-mutants of potato. It was than surprisingly found that amf-mutants,resulting from crosses with wild-type potatoes had increased storagecapacity for proteins in their tubers, these days considered aneconomically desirable trait.

It is preferred that said high total raw protein content is alsoreflected in the amount of protein that can be harvested, e.g. from thetubers. Such measure is given by identifying the fraction of coagulatingprotein available for harvest, as further explained in the detaileddescription. The invention also provides a potato plant or part derivedthereof (such as a cell, a protoplast, a tuber, an embryo, a seed or anexplant) having at least one amf-gene said potato plant or part furtherprovided with an increased capacity to produce harvestable protein ascharacterized by a total coagulating protein content of its tubers(preferably as determined in the potato juice derived thereof) at least0.9%, more preferred at least 1.2%, most preferred at least 1.5%.Considering that high protein levels are these days often moreprofitable than high starch levels, the invention also provides a highprotein potato (i.e. with more than 1.2%, preferably more than 1.5%coagulating protein in its tubers) characterized in that its tubersessentially show a coagulating protein versus starch ratio of at least45 kg/ton, more preferred of at least 90 kg/ton.

Furthermore, the invention provides a high-protein potato according tothe invention characterized in that it is a transgenic potato, forexample provided with a gene or gene encoding for a heterologousprotein, for example with the purpose to provide a high protein potatoaccording to the invention additionally provided with increased levelsof essential amino acids. It is preferred that such a heterologousprotein comprises a heterologous protein rich in essential amino acids.About half of the 20 amino acids found in proteins can be made byvertebrates; the others must be supplied in the diet. For this reason,the latter are called essential ammo acids. These include the strictlyessential amino acids which are lysine, leucine, isoleucine, valine,phenylalanine, methionine, threonine and tryptophan. Additionally,tyrosine and cysteine, although they are not strictly essential, must beconsidered as such, since they are synthesised only from essential aminoacids: tyrosine from phenylalanine and cysteine from methionine. Inparticular, humans and other monogastric animals cannot synthesise theessential amino acids and need to obtain these from their diet. The dietof humans and livestock is largely based on plant material. However,several of these essential amino acids are often only present in lowconcentrations in crop plants, which mainly constitute said plant baseddiets. In particular, lysine, threonine, methionine or tryptophane oftenlack in such diets. Dietary proteins are often not nutritionallyequivalent, which correlates with the amino acid composition of thedifferent proteins. Feeding a diet that provides an inadequate amount ofone of the essential amino acids leads to negative nitrogen balance,since the normal catabolism of proteins continues, but new synthesis forreplacement is limited by the relative lack of the essential amino acid.This occurs even when the total dietary intake of protein is apparentlyadequate. The extent to which a dietary protein can be used for thesynthesis of tissue proteins is limited by the content of the essentialamino acid that is present in an amount relative to the requirement.This is the limiting amino acid of that protein. Now that a high proteinpotato is provided it is beneficial to age this for the expression andstorage of valuable proteins. The invention furthermore provides atransgenic potato cell with at least one amf-allele having been providedwith a nucleic acid encoding a proteinaceous substance, a sink protein.In a preferred embodiment, said cell accumulates said sink protein up tomore than 2%, preferably 4%, or even more than 5% to more than 7% of thetotal protein content of said cell. The protein preferably contains ahigh content of essential amino acids (preferably methionine, cysteine,lysine, threonine, or tryptophane). In a preferred embodiment, theinvention provides a high protein potato according to the inventioncomprising a heterologous protein rich in essential amino acids such aslisted in Table 4.

The invention also provides a method for breeding and selecting a potatowith an increased capacity to store a protein comprising crossing afirst parent potato with at least one amf-gene with a second parentpotato without an amf-allele, and selecting progeny for the presence ofat least one amf-allele and for a protein content of its tubers higherthan detected in said first parent or said second parent. It ispreferred that progeny is selected or a protein content of its tubershigher than detected in said first parent and said second parent. Ofcourse, the storage of proteins being most fully enhanced inamylose-free plants, it is most preferred to select progeny homozygousfor the amf-allele.

In a preferred embodiment, the invention provides a method for breedingand selecting a potato with an increased capacity to store a proteincomprising crossing a first parent potato with at least one amf-allelewith a second parent potato without an amf-gene, and selecting progeny(preferably homozygous for the amf-allele) by testing it or the presenceof at least one amf-gene and testing it for total raw protein contentwith a method, such as the Kjeldahl method as described herein, todetermine total raw protein content of its tubers and selecting progenywith a total raw protein content higher than detected in said firstparent or said second parent, said method preferably further comprisingselecting progeny with a total raw protein content of its tubers higherthan detected in said first parent and said second parent.

In a further preferred embodiment, the invention provides a method forbreeding and selecting a potato with an increased capacity to produceharvestable protein comprising crossing a first parent potato with atleast one amf-allele with a second parent potato without an amf-allele,and selecting progeny (preferably homozygous for the amf-allele) bytesting it for the presence of at least one amf-allele and testing itfor coagulating protein content with a method comprising determiningtotal raw protein and total soluble raw protein remaining in solutionafter a protein coagulation procedures, such as immersion in a boilingwater bath as described herein, to determine or calculate totalcoagulating protein content of its tubers and selecting progeny with atotal coagulating protein content higher than detected in said firstparent or said second parent, said method preferably further comprisingselecting progeny with a total coagulating protein content of its tubershigher than detected in said first parent and said second parent.

Furthermore, the invention provides a potato selected with a methodaccording to the invention, use of a potato as provided herein for theindustrial production of starch and/or protein and use of a potato asprovided herein in breeding and selection programmes of potatoes. Inparticular, the invention provides use of a potato plant or part derivedthereof having at least one amf-allele in a breeding and selectionprogramme directed at providing potatoes with an increased proteincontent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4.

Examples of breeding schemes for breeding and selecting potatoes

DETAILED DESCRIPTION OF THE INVENTION

Using an amylose-free (amf)mutant of diploid potato (Solanum tuberosum),diploid and tetraploid clones with different genotypes at the amf-locuswere produced. In order to make use of the diploid material in analyticbreeding of amf-potatoes, clones were selected that producedconsiderable frequencies of 2n-pollen and 2n-eggs. Successful attemptswere made to select normal synaptic as well as desynaptic clonesproducing 2n-gametes. When for example microspores are stained with apotassium iodide solution (Lugol), starch is stained red in mutants(comprising only the amf-gene) and dark blue in the wild type(comprising only the Amf-allele). Based on the phenotype of starch inthe microspores, tetraploid clones with nulliplex, simplex, duplex,triplex and quadriplex genotypes at the Amf-locus were selected. Weinvestigated starch properties and protein content in various parts ofthe mutant potato plant. Starch composition and protein content intubers turned out to be an easily scorable feature. It allows inbreeding programmes for amylose-free potatoes an early assessment ofstarch and/or protein composition in the prospective plants or partsthereof.

Plant material. Monoploid amylose-free (amf) clone 86.040 and the parentclone AM79.7322 are described in Hovenkamp-Hermelink et al. (1987,ibid). Doubled amf-plants were obtained by adventitious shootregeneration on leaf explants, which were taken from in nitro propagatedshoots of monoploid 86.040. After root induction in MS₃₀ (Murashige &Skoog, 1962, Physiol. Plant 15:473-497) (MS) medium supplemented with 30g/l sucrose) a number of these diploid amf-plants were transferred intoa glasshouse, at 19° C. at day: 17° C. at night and 16 h daylength, insterilized leaf containing soil. For better flowering, part of thedoubled plants was grafted onto tomato rootstock. Pollen fertility wasestimated, after aceto carmine staining. For the crosses, a variety ofwild-type potato pollen was used. The crosses were made on open flowersof diploid (2x) clones of 86.040. The wild-type potato clones had beenselected for good male an finale fertility and 2n-gametes (unreducedgamete) production in male and female parents.

This resulted in breeding material with better fertility both on maleand female side giving opportunity to make crosses with more advanceddiploid breeding material. From these crosses diploid as well astatraploid progeny (4x) was obtained. Diploid material was segregatingfor the amf-allele, resulting in 25% homozygous plants, which could beselected by colouring the tubers with a iodine solution. Also sometetraploid progeny could be obtained as a result of unreduced pollen andunreduced eggcells in both parents. This bilateral sexualpolyploidiation has also been used in a third cycle of crosses, makinguse of diploid homozygous amf-clones. A second method to achievetetraploid progeny has been 4x.2x crosses, where only the pollendonorhas to form unreduced gametes to get a tetraploid progeny (unilateralsexual polyploidization).

In general the first two or three cycles of this breeding program havebeen used to produce male and female fertile amf-breeding material, onthe diploid level with the ability to produce unreduced gametes, as astart for a breeding program on diploid and tetraploid chromosomallevel. In these breeding cycles also properties as tuber shape, numberof tubers and starch content where observed, but no stringent selectionwhere carried out.

From the third cycle onwards crosses have been made between homozygoustetraploid amf clones with existing tetraploid starch potato varieties.From these varieties genetic variation with respect to total starchproduction and resistance against diseases (potato cyst nematode, lateblight, wart disease) was introduced. In the second cycle of thesecrosses made for agronomic improvement segregation of homozygousrecessive amf-clones was expected and colouring of tubers with iodinesolution was carried out. As a result of this breeding program someagronomical acceptable clones were produced, which are both useful forthe large scale production of amylosefree potato starch and as crossingparents in 4x.4x and 4x.2x crosses.

Starch analysis. Starch granules in micropores and tubers were stainedwith I₂-KI solution according to Hovenkamp-Hermelink et al. (1987), instomatal guard cells and other leaf cells according to the treatmentdescribed for, microspores and in root cap cells by treatment of roottips with a mixture of Lugols-solution and choralhydrate (1:1, v/v).Four gram of choralhydrate is dissolved in 2 ml of water. The amylosepercentage in starch solutions of tubers was measured according toHovenkamp-Hermelink et al., 1988, Potato Res. 31:241-246). Roottips werefixed and stained according to Pijnacker and Ferwerda (1985, Can. J.Genet. Cytol. 26:415-419) for chromosome counts and karyotypicinvestigations. When for example microspores are stained with apotassium iodide solution (Lugol), starch is stained red in mutants(comprising only the amf-gene) and dark blue in the wild type(comprising only the Amf-gene) (Jacobsen et al., 1989, Euphytica44:43-48).

Protein Analysis

For determining raw and coagulated protein content, 300 grams of tubermaterial together with 1000 ppm sodium bisulphite was grinded in alaboratory blender, type Waring Blendor. To determine the dry mattercontent an homogeneous sample of approx. 10 gram was taken and driedovernight at 40° C. The rest of sample was centrifuged for 10 min at4600 rpm. Of the supernatant raw protein content was determined bydetermining nitrogen content with the Kjeldahl method and dry matter byovernight drying at 40° C. To determine the coagulated protein contentin the supernatant the pH was adjusted to 5.2 with 19% HCl and theliquid was boiled for 1 minute. Subsequently, the samples werecentrifuged for 10 ml at 10000 rpm. To remove the light substance theabove liquid was filtered over an S&S 595 paper filter. Nitrogen contentof the supernatant after the coagulation step was determined by theKjeldahl method. All experiments were carried out in duplicate.

Raw and coagulated protein content was calculated as follows:

${{Contribution}\mspace{14mu}{of}\mspace{14mu}{juice}} = \frac{100 - {\%\mspace{14mu}{DrySubstancePulp}}}{100 - {\%\mspace{14mu}{DrySubstanceJuice}}}$Raw protein=N total×contribution juice×1.5×0.88×6.25Coagulated protein=(N total−N after coagulation)×6.25×contributionjuice×1.5×0.88(1.5: dilution factor)(0.88: correction factor)

Embryo culture. Unripe berries were surface sterilized by treatment for1 minute with 70% alcohol and for 15 minutes with a saturated solutionof Ca-hypochlorite, containing a few drops of 1% SDS (sodiumdodecylsulphate) solution per 100 ml. The sterilized berries were cutopen aseptically. Ovules were collected and cultured on medium EC2(MS-medium supplemented with 1.10⁻⁶ g/l kinetin, 1.10⁻⁶ g/l LAA, 8 g/lagar and 30 g/l sucrose) as defined by Neal & Topoleski (1983, J. Amer.Soc. Hort. Sci. 108:434-448; 1985 J. Amer. Soc. Hort. Sci. 110:869-873)for embryo culture of tomato. During ovule culture, the integumentrapidly attained a brow color and was removed; this was followed laterby entire excision of the embryo from the endosperm, as described byHaynes (1959). The excised embryos were also cultured on medium EC2, at23° C. and 16 h light. The rescued plantlets were propagated and rootedin MS₂₀.

Results

Identification of Amf-Gene Mutants

Based on iodine staining of microspores, genotypes corresponding totnulliplex (no wild-type GBSS-allele), simplex, duplex andtriplex/quadruplex for the wild-type GBSS allele were selected. Thisselection was according to the expected segregation presented in Table1.

TABLE 1 The expected and obtained offspring when duplex plants (AAaa ×AAaa) are crossed. These genotypes can be distinguished after iodinestaining by their segregation of blue and red microspores; triplex(AAAa) and quadruplex (AAAA) plants where taken in one group. Genotypeswith enough tubers to perform a field trial were selected. microsporenumber of number of plant segregation genotypes gynotypes genotypechance blue:red found^(a) selected aaaa 1/36 0:1  3  2 Aaaa 8/36 1:1 2010 AAaa 18/36  5:1 33 11 AAAa 8/36 1:0 19  6 AAAA 1/36 1:0 a: χ² (1:8;18:9) = 1.62 < 7.82 which indicates that the offspring is not deviatingfrom the expected 1:8:18:9 segregation of the gene-dosage genotypes forthe wild-type GBSS allele.

Starch granules of the duplex and triplex/quadruplex genotypes werecompletely blue. In some of the simplex genotypes however, a small outerlayer was red in a small percentage of the starch granules. A number oftuberising plants belonging to each gene-dosage group was selected forfurther research in a field trial Table 1).

TABLE 2 The coagulated protein content analysis of offspring when duplexplants (HZ91-RUG-025 × HZ91-RUG-075) are crossed. These genotypes weredistinguished after iodine staining by their segregation of blue and redmicrospores; triplex (AAAa) and quadruplex (AAAA) plants where taken inone group. plant genotype No. individuals mean S. E. aaaa 15 1.51** 0.08Aaaa 15 1.22 0.06 AAaa 17 1.10 0.12 AAAa 25 1.24 0.07 AAAA **indicatesstatistically significant effect P < 0.05

TABLE 3 The coagulated protein content analysis of offspring when duplexplants (S90-1103 × S90-1101-0004) are crossed. These genotypes weredistinguished after iodine staining by their segregation of blue and redmicrospores; triplex (AAAa) and quadruplex (AAAA) plants where taken inone group. plant genotype No. individuals mean S. E. aaaa  8 1.67** 0.08Aaaa 18 1.38 0.07 AAaa 17 1.25 0.12 AAAa 29 1.46 0.03 AAAA **indicatesstatistically significant effect P < 0.05GBSS-Protein Content

The amount of GBSS-protein in the starch granule of different genotypeswas analyzed. FIG. 1 clearly shows that the amylose-free plants had noGBSS in the starch granules, however no significant difference could beobserved in the GBSS-protein level of the other groups indicating thatno dosage effect existed at the protein level. No differences in starchgranule size and amylopectin and sucrose content of the tubers werefound (data no shown)

Overexpression of Heterologous Protein in amf Mutant

The invention furthermore provides a transgenic potato cell with atleast one amf-gene having been provided with a nucleic acid encoding aproteinaceous substance, a sin protein. In a preferred embodiment, saidcell accumulates said sink protein up to more than 2%, preferably 4%, oreven more than 5% to more than 7% of the total protein content of saidcell. The protein preferably contains a high content of essential aminoacids preferably methionine, cysteine, lysine, threonine, ortryptophane). To allow for an enhanced incorporation of these essentialamino acids into a sink protein fraction of the amf potato cell saidcell is provided with one or more gene constructs or nucleic acidmolecules encoding at least one functional enzyme related to said aminoacid's biosynthesis pathway allowing said cell to increasinglysynthesise said amino acid, preferably wherein said amino acid is anessential amino acid and thereby further regulates supply. Preferably,free amino acid level is increased by introducing at least one geneencoding a feedback insensitive enzyme involved in biosynthesis of saidamino acid. The over-produced free essential amino acids are trapped byincorporation in a sink protein, rich in said essential amino acid thatis expressed at the same time in the plant.

As food or feed organisms, or tissues, differ in limiting essentialamino acids, the optimal amino acid content for a sink protein variesaccording to organism. A sink protein preferably is a proteinspecifically enriched in those amino acids for which a definite occursin the specific crop or organism. By producing the sink protein to atleast 2%, preferably to at least 4%, 5%, or even at least 7% of thetotal protein content of the tissue which is being used as food or feed,we compensate for the essential limiting amino acid. For example, forpotato a sink protein preferably contains at least 5%, more preferablyat least 10% lysine, at least 2.5% methionine, at least 2.5% cysteine,or at least 1.6% tryptophan.

The protein is stable in the plant, accumulates to high levels and hasno drastic detrimental effects on the growth and physiology of the cropplant. The protein is well digestible by the livestock and/or humandigestive tract.

Sink protein candidates can for example be selected from among knownstorage proteins. Several publications describe the amino acidcomposition of plant storage proteins, and their possible use to enhancethe essential amino acids composition of food and feed crops. Thestorage proteins of cereal crops like wheat, barley and maize of theso-called prolamin type vary in their content of sulphur-containingamino acids (methionine and cysteine). Some are relatively high inS-rich amino acids. However, most of them are severely deficient inlysine and tryptophan (Shewry, P. R., (1998) Transgenic Plant Research,p. 138-149). The storage proteins in legumes and other dicotyledons aremainly of the globulin family or the albumin family. Globulins aregenerally very poor in the sulphur containing amino acids, but sometimesdo contain a relatively high ratio of lysine. Vicilin (of Vica faba) hasa lysine content of 7.2%, threonine content of 3% and a methioninecontent of only 0.2%. The 2S albumin family of storage proteins ingeneral have a high content of S-rich amino acids. Brazil nut 2S albumincontains ca. 26% sulfur amino acids (Ampe 1986), and sunflower 2Salbumin (Sfa8) contains 24% sulfur amino acids (Kortt 1991). Otherstorage proteins that have a high content of lysine residues are theprotease inhibitors C1 and C2 from barley (9.5% and 11.6% lysinerespectively, Hejgaard and Boisen (1980)) and the cysteine proteaseinhibitor multicystatin of potato (Waldron et al., 1993 Plant Mol. Biol.23(4):801-12).

The level of protein accumulation in a plant is determined by the rateof synthesis in relation to the rate of degradation of this protein. Therate of degradation is determined by its sensitivity to attack ofproteases that are present in the producing tissue. This proteasesensitivity is influenced by the availability of susceptible sequencedomains on the surface of the protein, in combination with thestructural rigidity of the protein. In order to select for a proteinthat will have a high chance to accumulate in the plant, the proteinpreferably has a rigid tertiary structure, with minimal exposed sequencedomains. Certain proteins have a native tendency to aggregate into moreor less regular or organized macromolecular structures, such as proteinbodies or protein crystals. Naturally, storage proteins that accumulatein plant tissue, where they have a storage function, are naturallyadapted to remain stable in these plant tissues. Therefore seed storageproteins are distinct candidates to accumulate essential amino acids.However, few plant storage proteins will always have a desiredcomposition relating to the desired essential amino acids. Furthermore,in general the amount of essential amino acids is often too low. Theinvention herewith provides using sink protein that is encoded by anucleic acid enriched with the necessary codons encoding said desiredamino acids. In addition to this, in nature several proteins exist thatform (semi)-crystaline structures in their natural tissue. Examples aresome peroxisomal proteins like alcohol oxidase or urate oxidase, orcrystallins (eye lens proteins). Also plant structural proteins are ableto form regular crystal like structures, for example the cysteineprotease inhibitor multicystatin present in the peel of potato.

As an example the use of a combination of gene constructs containing aDNA sequence encoding an enzyme having dyhydrodipicolinate synthase(DHPS) activity combined with a DNA sequence encoding a sink proteinthat is rich in essential amino acids, e.g. multicystatin, is provided.First half of this construct containing the DHPS genes results in anincreased level of free lysine more than 10-fold the wild type level ofeach amino acid in a plant or parts thereof. The expression regulationshould be such that expression occurs in such a way that lysine areproduced to a comparable extent without damaging the plant i.e. withoutcausing negative aberrations in the phenotype compared to wild typeplants. The second part of this combination of gene constructs consistsof a gene encoding a sink protein, which contains a high amount ofessential amino acids. This sink protein results in an increasedincorporation of essential amino acids into the protein fraction. Assuch it withdraws these amino acids from the pool of free amino acids,thus further enhancing the synthesis of these essential amino acids.

Example 1 Chimeric Gene Construct with the Mutant Potato DHPS Gene

DNA isolation, subcloning, restriction analysis and DNA sequenceanalysis is performed using standard methods (Sambrook, J. et al. (1989)Molecular Cloning. A laboratory manual, Cold Spring Harbor LaboratoryPress; Ausubel, F. M. et al. (1994) Current protocols in molecularbiology, John Wiley & Sons).

In order to create a feedback insensitive DHPS, the evolutionaryconserved amino acid residue 134 (asparagine) were changed into acysteine residue (WO0148230). The mutant DHPS encoding DNA fragment(designated DEPS-134nc1) was used for the expression in potato plants.

The chimeric gene containing the mutant DHPS gene was constructed bysubcloning DHPS cDNA from the pTriplex vector in pCR-cript SK(+) andfrom this vector as a XbaI-Eco RI fragment in the pBluescript SE vectordigested with XbaI-EcoR. With this clone the mutagenesis was performed,resulting in clone pAAP57-134nc1. At the 5′ end the mutated DHPS cDNAwas fused to a HindIII-SalI fragment of the 800 bp long GBSS promoterfragment (Visser et al. ibid). Downstream of the mutant DHPS sequencethe termination signal of the nopaline synthase gene from Agrobacteriumtumefaciens was inserted (Greve, H. D. et al. (1983) J. Mol. Appl.Genet. 1: 499-511) as an SstI-EcoRI fragment. The complete chimeric genewas subcloned into the HindII-EcoRI sites of pBINPLUS (Van Engelen, F.A. et al. (1995) Transgenic Research 4: 288-290) (pAAP105).

The binary vector pAAP105 was used for freeze-thaw transformation ofAgrobacterium tumefaciens strain AGLO (Höfgen, R. and Willmitzer, L.(1988) Nucl. Acids Res. 16: 9877). Transformed AGLO was subsequentlyused for inoculation of potato (Solanum tuberosum, variety Kardal) stemexplants as described by Visser (Visser, R. G. F. (1991) Plant TissueCulture Manual B5 (ed. by K. Lindsey): 1-9, Kluwer Acad. Publishers, TheNetherlands). After shoot and root regeneration on kanamycin-containingmedia plants were put in soil and transferred to the greenhouse. Plantsregenerated (on kanamycin-free media) from stem explants treated withthe Agrobacterium strain AGLO lacking a binary vector served as acontrol.

Example 2 Overexpression of the Potato Multicystatin Gene

The Potato Multicystatin (hereinafter PMC) gene encodes a multidomaincysteine protease inhibitor protein. A genomic clone of the PMC gene(Waldron et al., (1993) Plant Molecular Biology, 23:801-812) was fusedat the 5′ end to the omega DNA sequence from the coat protein of tobaccomosaic virus (Gallie, D. R. et al. (1987) Nucl. Acids Res. 15:3257-3273). Downstream of the PMC sequences the termination signal ofthe octopine synthase gene from Agrobacterium tumefaciens is inserted(Greve, H. D. et al. (1988) J. Mol. Appl. Genet. 1: 499-511). Thechimeric PMC gene construct is cloned as a BamHI/SpeI fragment inpBluescript. The patatin promoter (Wenzler, H. C. et al. (1989) PlantMol. Biol. 12: 41-50) is ligated as a blunt (HindIII filled in)/BamHIfragment in front of the PMC chimeric gene digested with SmaI/BamHI(pAAP169).

Example 3 Transformation of Potato Plants

The binary vector pAAP105 and PAAP169 is used for freeze-thawtransformation of Agrobacterium tumefaciens strain AGLO (Hofgen, R. andWillmitzer, L. (1988) Nucl. Acids Res. 16: 9877). Transformed AGLO issubsequently used for inoculation of tetraploid wildtype potato (Solanumtuberosum, variety Kardal and tetraploid amf mutant KA96-1396 stemexplants as described by Visser (Visser, R. G. F. (1991) Plant TissueCulture Manual B5 (ed. by K. Lindsey): 1-9, Kluwer Acad. Publishers, TheNetherlands). After shoot and root regeneration on kanamycin-containingmedia plants are put in soil and transferred to the greenhouse. Plantsregenerated (on kanamycin-free media) from stem explants treated withthe Agrobacterium strain AGLO lacking a binary vector serve as acontrol.

Example 4 Analysis of Free No Acid Content in Transgenic Plants

Tissue (0.5-1.0 gram) is homogenized with mortar and pestle in 2 ml 50mM Pi-buffer (pH 7.0) containing 1 mM dithiothreitol. Nor-leucine isadded as an internal standard. Free amino acids are partly purified byextraction with 5 ml of a water:chloroform:methanol mixture (3:5:12).Water phase is collected and the remaining re-extracted twice. Afterconcentration by lyophilization to 3 ml, a 20 mu l sample is analysed byHPLC using a cation-exchange column with post-column ninhydrinederivatisation of the amino acids detected at 570 and 440 nm (BIOCHROM20, Amersham Pharmacia biotech).

Example 5 Coagulated Protein Content of Transgenic Plants

For determining raw and coagulated protein content, 300 grams of tubermaterial together with 1000 ppm sodium bisulphite was grinded in alaboratory blender, type Waring Blendor. To determine the dry mattercontent a homogeneous sample of approx. 10 gram was taken and driedovernight at 40° C. The rest of sample was centrifuged for 10 min at4600 rpm. Of the supernatant raw protein content was determined bydetermining nitrogen content with the Kjeldahl method and dry matter byovernight drying at 40° C. To determine the coagulated protein contentin the supernatant the pH was adjusted to 5.2 with 19% HCl and theliquid was boiled for 1 minute. Subsequently, the samples werecentrifuged for 10 min at 10000 rpm. To remove the light substance theabove liquid was filtered over an S&S 595 paper filter. Nitrogen contentof the supernatant after the coagulation step was determined by theKjeldahl method. All experiments were carried out in duplicate.

TABLE 4 LYSINE RICH Vicilin Fava bean 436 aa 32 lys (7.2%) storageprotein SCR1 Soybean 102 aa 21 lys (20.6%) stress induced Fcor 2Strawberry 133 aa 19 lys (14.3% cold induced TLRP Tomato  62 aa 11 lys(17.7%) matrix protein multicystatine Potato 11.8% lys Proteaseinhibitor METHIONINE RICH γZein Maize 211 aa 55 met (26.1%) storageprotein 10 kDa Zein 150 aa 31 met (20.7%) storage protein 2S albuminSunflower 141 aa 18 met (12,8%) storage protein THREONINE RICH TIP13Asparagus 182 aa 23 thr (12.6%) harvest PTGRP Tomato  78 aa 16 thr(20.5%) water stress CYSTEINE RICH PA1b Pea 130 aa 10 cys (7.7%) storageprotein SE60 Soybean  47 aa  8 cys (17.2%) storage protein PCP1 RapeSeed  83aa  8 cys (9,6%) pollen/stigma

1. A method for breeding and selecting a potato having increased proteincontent comprising (a) crossing a first parent potato plant having atleast one amf-allele with a second parent potato plant having at leastone amf-allele to produce progeny; (b) selecting and testing saidprogeny for the presence of at least one amf-allele and for increasedprotein content; and (c) selecting progeny being homozygous for theamf-allele with a protein content higher than a plant heterozygous forthe amf-allele.
 2. A method for increasing protein storage in a potatocomprising (a) crossing a first parent potato plant having at least oneamf-allele with a second parent potato plant having at least oneamf-allele to produce progeny; (b) selecting and testing said progenyfor the presence of at least one amf-allele and for increased proteincontent; and (c) selecting progeny being homozygous for the amf-allelewith protein content higher than a plant heterozygous for theamf-allele.
 3. The method according to claim 2, wherein the proteincontent of tubers of the selected progeny is at least 0.9% m/m.
 4. Themethod according to claim 3, wherein the protein content of tubers ofthe selected progeny is at least 1.2% m/m.
 5. The method according toclaim 4, wherein the protein content of tubers of the selected progenyis at least 1.5% m/m.
 6. The method according to claim 2, whereincoagulating protein versus starch ratio of the selected progeny is atleast 45 kg/ton.
 7. The method according to claim 6, wherein coagulatingprotein versus starch ratio of the selected progeny is at least 90kg/ton.
 8. The method according to claim 2, further comprisingtransforming said selected progeny with a gene encoding a heterologousprotein.
 9. The method according to claim 8, wherein the heterologousprotein is selected from the group consisting of DHPS, PMC, vicilin,SCR1, Fcor2, TLRP, multicystatine, yZein, 10 kDa Zein, 2S albumin,TIP13, PTGRP, PA1b, SE60 and PCP1.